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. 2015 Mar 17;11(3):e1004725.
doi: 10.1371/journal.ppat.1004725. eCollection 2015 Mar.

Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape

Jens Dinter  1 , Ellen Duong  1 , Nicole Y Lai  1 , Matthew J Berberich  1 , Georgio Kourjian  1 , Edith Bracho-Sanchez  1 , Duong Chu  1 , Hang Su  1 , Shao Chong Zhang  1 , Sylvie Le Gall  1
Affiliations

Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape

Jens Dinter et al. PLoS Pathog. .

Abstract

Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The immunodominant HLA-B57-restricted TW10 and KF11 epitopes are more efficiently cross-presented than subdominant ISW9 epitope.
A. Immature DCs (open symbols) and Møs (solid symbols) were incubated with recombinant HIV-1 p24 protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with HLA-B57 ISW9-specific (☐), KF11-specific (○), and TW10-specific (Δ) CTL clones as effector cells. CTL responses in form of spot forming cells are shown for n≥10 donors. B. CTL responses to immature DCs (open symbols) and Møs (solid symbols) pulsed with different concentrations of optimal epitope B57-ISW9 (☐) or B57-TW10 (Δ) were measured in an overnight IFN-gamma ELISPOT assay. Data are representative of two independent experiments with different donors. Due to the limited number of DCs and Møs generated from HLA-B57+ donors, the titration of epitopes was only done for B57-ISW9 and B57-TW10. C. Immature DCs (open bars) and immature Møs (solid bars) were pre-treated with a cocktail of protease inhibitors to reduce intracellular degradation before addition of different concentrations of recombinant HIV-1 p24 protein at 37°C or 4°C. Internalized p24 protein was determined by a standard p24 ELISA assay using whole cell extracts from lysed DCs and Møs. Results are from three independent experiments with different donors and show mean ± SD. D. Immature DCs (open bars) or immature Møs (solid bars) were incubated with different concentrations of recombinant HIV-1 p24 protein and used as antigen presenting cells to B57-KF11-specific CTLs. Responses in form of spot forming cells are shown. Data are representative of two independent experiments with different donors.
Fig 2
Fig 2. Inhibition of proteasomes increases B57-KF11 and B57-ISW9 epitope cross-presentation by DCs but not Møs (A) Immature DCs (open symbols) and (B) immature Møs (solid symbols) were pretreated with proteasome inhibitor (MG132) or a broad inhibitor of cysteine proteases (E64) before addition of recombinant HIV-1 p24 protein.
CTL responses to HLA-B57 ISW9-specific (☐), KF11-specific (○), and TW10-specific (Δ) were measured as described in Fig. 1. Similarly, incubation of DCs with epoxomicin, a selective chymotrytpic proteasome inhibitor, increased HLA-B57-KF11-specific CTL responses by 11-fold. Results are from n≥5 donors.
Fig 3
Fig 3. The immunodominant epitope B57-KF11 is more efficiently produced in cross-presentation-competent compartments than subdominant epitope B57-ISW9.
A. Two nmol of p24–35mer (MVHQAISPRTLNAWVKVVEEKAFSPEVIPMFAALS, aa 10–44 in Gag p24) were degraded in 15μg of whole cell extracts from immature and mature DCs and Møs for 10, 30, 60, and 120 minutes in degradation buffer at pH7.4, pH5.5 or pH4.0. Degradation products identified by mass spectrometry were grouped according to their lengths of fragments: equal or longer than 26 aa (blue), 19–25 aa (orange), 13–18 aa (gray), 8–12 aa (red), and fragments equal or shorter than 7 aa (light gray). The peak area of each identified peptide was calculated with Proteome Discoverer and the contribution of each category of peptides to the total intensity of all degradation products is shown at each time point. B. All degradation products of p24–35mer were identified as described in (A). Peptides were grouped into fragments containing B57-ISW9 and B57-KF11 epitopes (black), containing only B57-KF11 epitope (red), containing only B57-ISW9 epitope (blue), or neither epitope (gray), respectively. C. Cleavage patterns of p24–35mer incubated with whole cell extracts from immature DCs for 30 minutes (upper panel) or 120 minutes (lower panel) at pH7.4, pH5.5, and pH4.0 are shown as the contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a specific amino acid, to the total intensity of all degradation products. For (A-C) data are representative of three independent experiments with three different donors.
Fig 4
Fig 4. Slow degradation of TW10-containing peptides results in high amounts of B57-TW10 available for cross-presentation.
A. Two nmol of p24–31mer (GSDIAGTTSTLQEQIGWMTNNPPIPVGGEIY, aa 101–131 in Gag p24) were degraded in 15μg of whole cell extracts from immature and mature DCs and Møs for 10, 30, 60, and 120 minutes in degradation buffer at pH7.4, pH5.5 or pH4.0. Degradation products identified by mass spectrometry were grouped according to their lengths of fragments as described in Fig. 3. The contribution of each category of peptides to the total intensity of all degradation products is shown at each time point. B. All degradation products of p24–31mer were grouped into fragments containing B57-TW10 (black), B57-TW10 epitope with N-terminal extensions (white), B57-TW10 epitope with C-terminal extensions (red), B57-TW10 epitope with N- and C-terminal extensions (green), antitopes defined as fragments lacking B57-TW10 (blue), or the original peptide (gray), respectively. The contribution of each category of peptides to the total intensity of all degradation products is shown at each time point.
Fig 5
Fig 5. Limited degradation of RK9-containing fragments results in cross-presentation of high amounts of HLA-A03 RK9 epitope.
A. Immature DCs (◇) or Møs (◆) were incubated with recombinant HIV-1 p55 protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with HLA-A03 RK9-specific CTLs as effector cells. CTL responses in form of spot forming cells are shown for n≥5 donors. B. CTL responses to immature DCs (◇) and immature Møs (◆) pulsed with different concentrations of optimal epitope A03-RK9 were measured as described before. C. Immature DCs (open bars) or Møs (solid bars) were incubated with different concentrations of recombinant HIV-1 p55 protein and used as antigen presenting cells in an overnight IFN-gamma ELISPOT assay with HLA-A03 RK9-specific CTLs as effector cells. CTL responses in form of spot forming cells are shown. D. Cleavage patterns of p17–17mer incubated with whole cell extracts from immature DCs for 120 minutes at pH7.4, pH5.5, and pH4.0 are shown as the contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a specific amino acid, to the total intensity of all degradation products. For (B-D) data are representative of two independent experiments with different donors.
Fig 6
Fig 6. The intracellular stability of HIV-1 epitopes in the cytosol and lysosomes follows CTL responses hierarchy.
A. One nmol of highly purified HLA-B57-restricted KF11 (○) and HLA-A03-restricted RK9 (◇) were degraded in 15μg of immature DC or Mø extracts (open or solid symbols, respectively) in degradation buffer at pH7.4 (solid line) or pH4.0 (dashed line). Degradation products were analyzed by RP-HPLC after 10, 30, and 60 minutes. 100% represents the amount of peptide detected at time 0, calculated as the surface area under the peptide peak. B. The stability rate of optimal epitopes B57-ISW9, B57-KF11, B57-FF9, B57-TW10, A11-ATK9, A03-RK9, and A03-KK9 at pH7.4 and pH4.0 was calculated by a nonlinear regression (one-phase exponential decay) of the degradation profile obtained over a 60-minute incubation in extracts of immature and mature DCs (upper panel) and Møs (lower panel). Bars represent the mean ± SD of three independent experiments for each epitope with extracts from different healthy donors.
Fig 7
Fig 7. Escape mutations in immunodominant epitopes reduce epitope production and stability in the cross-presentation-competent cell compartments.
A. Two nmol of 5-TW10–3 (DIAGTTSTLQEQIGWMTN, aa 103–120 in Gag p24), 5-TW10–3 T3N (DIAGTTSNLQEQIGWMTN), and 5-TW10–3 T3N/G9A (DIAGTTSNLQEQIAWMTN) were degraded in 15μg of whole cell extracts from immature DCs for 10, 30 or 60 minutes in degradation buffer at pH7.4 and pH4.0. Degradation products identified by mass spectrometry were grouped into fragments containing the optimal epitope TW10 or its mutant (black), the epitope/mutant with N-terminal extensions (white), the epitope/mutant with C-terminal extensions (red), the epitope/mutant with N- and C-terminal extensions (green), antitopes defined as fragments lacking part of the epitope/mutant (blue), or the original peptide (gray), respectively. The contribution of each category of peptides to the total intensity of all degradation products is shown at each time point. B. Cleavage patterns of 5-TW10–3, 5-TW10–3 T3N, and 5-TW10–3 T3N/G9A incubated with whole cell extracts from immature DCs for 10 minutes at pH7.4 (upper panel) or at pH4.0 (lower panel) are shown as the contribution of each cleavage site, presented as cleavage N-terminal or C-terminal to a specific amino acid, to the total intensity of all degradation products. For (A-B) data are representative of two independent experiments with different donors. C. One nmol of highly purified HLA-B57-restricted TW10, TW10-T3N or TW10-T3N/G9A mutants were degraded in 15μg of immature DCs or Møs extracts (right and left panel) in degradation buffer at pH7.4 or pH4.0. A stability rate was calculated as described before. Bars represent the mean ± SD of three independent experiments for each epitope with extracts from different healthy donors.
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